1) Background The correct targeting and localization of transmembrane proteins is an essential aspect of cellular organization, particularly in large cells such as muscle which contains distinct membrane domains. The Golgi complex is the subcellular organelle responsible for this task. In muscle, the organization of the Golgi complex changes dramatically during differentiation and regeneration. Very little is known of the mechanism of these changes. The organization of the Golgi complex of muscle also depends on the pattern of contractile activity and this regulation is not understood at all. Our goal is to understand how the distribution of the Golgi complex in muscle is regulated and is linked to the functional needs of muscle. In the past we have uncoverered basic aspects of the changes that take place during differentiation. We have established that the Golgi complex of each myoblast fragments into hundreds of smaller independent Golgi complexes placed around the nuclei and throughout the cytoplasm of the multinucleated myotubes and muscle fibers. These small Golgi elements are not distributed randomly but are retained next to endoplasmic reticulum sites specialized in the export of proteins to the Golgi complex. This specific localization suggested that the fragmentation of the Golgi complex during muscle differentiation resembles the fragmentation that the Golgi complex undergoes when microtubules are depolymerized. We have indeed demonstrated the similarity between these processes, therefore identifying changes in the microtubule cytoskeleton as a key factor in the changes that take place during differentiation. Patterned activity is an important regulator of muscle metabolism and contraction but it had never been linked to the organization of the protein secretory pathway. We have discovered that the distribution of Golgi complex, endoplasmic reticulum exit sites and microtubules is plastic in mature muscle fibers and responds to the pattern of contractile activity, causing a fiber type-dependent organization. We hypothesize that this plasticity is important and allows muscle to fulfill different metabolic demands depending on patterned activity. 2) Objective of present studies Our past work gives, for the first time, a description of the organization of the Golgi complex in muscle. In order to understand how it is controlled by factors such as patterned activity, we need to determine which of the several vesicular and cytoskeletal elements involved is the trigger that organizes the others. For example, it is fundamental to determine whether microtubule changes cause the reorganization of the endoplasmic reticulum exit sites during differentiation or whether the two take place independently. It is also important to identify whether other cytoskeletal elements, besides microtubules, are involved in the redistribution of the Golgi complex. Recent results by others, for example, suggest that the desmin intermediate filament network might play a role and this is a possibility we need to explore. 3) Results during the past year A large effort has been geared towards the observation of endoplasmic reticulum exit sites in live cells as a function of microtubule status. This has been done by the simultaneous observation of fluorescent constructs of two different colors in the C2 mouse muscle cell line, together with treatment with pharmacological agents that disrupt microtubules. The results are consistent, so far, with a model in which microtubules organize the ER exit sites by acting on the ER itself. We have probed the role of the desmin intermediate filaments by studying the distribution of the Golgi complex proteins and associated cellular elements in muscle fibers of desmin-null mice (provided by Dr. Capetanaki, Baylor College of Medicine). The Golgi complex of desmin-null fibers is perturbed near the surface of the fibers but apparently normal in their core. Interestingly, we find that microtubules as well are perturbed at the surface but not in the core of the desmin-null fibers. All our results, so far, thus point to the primary importance of microtubules as regulators of the Golgi complex distribution in muscle. It becomes then important to understand how microtubules themselves are organized. An investigation of the changes in microtubule nucleation during differentiation is showing that nucleation, in differentiated muscle, takes place at three types of sites which differ from the myoblast centrosomes, although all seem to involve the protein gamma-tubulin. Therefore, new modes of recruitment of g-tubulin from a cytoplasmic pool may be responsible for the changes in microtubule organization during differentiation. 4) Conclusions and significance Cell biologists have been debating the relationship between Golgi complex and endoplasmic reticulum for several years. Our results suggest that in differentiated muscle the Golgi complex is tightly linked to the ER. The small but fully functional Golgi elements may indeed be viewed as appendages to the ER rather than independent organelles. This discovery may help us to understand what is happening in some pathological conditions, such as in neurodegenerative diseases, in which fragmentation of the Golgi complex has been suggested to contribute to the disease. Microtubules are essential integrators and regulators of subcellular architecture. Our results suggest that they may be play another important role as sensors of patterned contractile activity. The observation of microtubule defects in desmin-null mice also raises the possibility that microtubules may contribute to the muscle pathology of these mice.